This application claims the priority JP 2000-021842, filed Jan. 26, 2000.
The present invention relates to a method for separating and collecting nucleic acids, which is used for nucleotide sequence determination of nucleic acids, DNA analysis and so forth.
As methods for separating and collecting nucleic acids, the subtraction method, the differential display method and so forth have conventionally been used. Further, Japanese Patent Application Laid-open (Kokai) No. 4-325092 discloses a method for simultaneously collecting two or more kinds of nucleic acids by immobilizing single-stranded nucleic acids having different nucleotide sequences on separable multiple supports respectively, and bringing a sample nucleic acid mixture into contact with the supports to allow hybridization of each immobilized nucleic acid and a single-stranded nucleic acid in the mixture, having a nucleotide sequence complementary to the immobilized nucleic acid.
However, the method described in Japanese Patent Application Laid-open No. 4-325092 suffers from a problem that, since the nucleic acids are separated by separating the supports, kinds of usable supports are extremely limited. Further, since the supports must be separated in order to separate the nucleic acids, the method also suffers from a problem that it is difficult to simultaneously separate many kinds of nucleic acids. Moreover, the method suffers from a problem that separation of many kinds of nucleic acids requires a large amount of sample nucleic acid mixture, and the method also becomes troublesome.
The present invention was accomplished in view of the aforementioned problems, and an object thereof is to provide a method for separating and collecting nucleic acids that can separate and collect two or more kinds of nucleic acids in a simple manner.
The inventors of the present invention found that if hybridization was performed by using a substrate on which multiple kinds of nucleic acids are immobilized, and the hybridized nucleic acids were each separated and collected directly from the substrate without disassembling the substrate, the nucleic acids can be separated and collected in a simple manner, and thereby the aforementioned object can be achieved. Thus, they accomplished the present invention.
That is, the present invention provides the following.
(1) A method for collecting nucleic acids, which comprises:
a step of bringing a sample nucleic acid solution into contact with a nucleic acid-immobilized substrate comprising a substrate and two or more kinds of single-stranded nucleic acids separately immobilized on the substrate, to allow hybridization of the immobilized single-stranded nucleic acids and single-stranded nucleic acids complementary to the immobilized single-stranded nucleic acids, and
a step of collecting each of the hybridized single-stranded nucleic acids separately according to immobilized portions of the immobilized nucleic acids, without disassembling the nucleic acid-immobilized substrate.
(2) The method according to (1), wherein the nucleic acid-immobilized substrate is a substrate carrying a compound having a carbodiimide group.
(3) The method according to (1) or (2), wherein the nucleic acid-immobilized substrate is a DNA microarray.
(4) The method according to any one of (1) to (3), wherein the substrate has a plate-like shape.